Abstract

Predisposition to prostate cancer has a
genetic component, and there are reports of familial clustering of
breast and prostate cancer. Two highly penetrant genes that predispose
individuals to breast cancer (BRCA1 and
BRCA2) are known to confer an increased risk of prostate
cancer of about 3-fold and 7-fold, respectively, in breast
cancer families. Blood DNA from affected individuals in 38 prostate
cancer clusters was analyzed for germ-line mutations in
BRCA1 and BRCA2 to assess the
contribution of each of these genes to familial prostate cancer.
Seventeen DNA samples were each from an affected individual in families
with three or more cases of prostate cancer at any age; 20 samples were
from one of affected sibling pairs where one was ≤67 years at
diagnosis. No germ-line mutations were found in BRCA1.
Two germ-line mutations in BRCA2 were found, and both
were seen in individuals whose age at diagnosis was very young (≤56
years) and who were members of an affected sibling pair. One is
a 4-bp deletion at base 6710 (exon 11) in a man who had prostate cancer
at 54 years, and the other is a 2-bp deletion at base 5531 (exon 11) in
a man who had prostate cancer at 56 years. In both cases, the wild-type
allele was lost in the patient’s prostate tumor at the
BRCA2 locus. However, intriguingly, in neither case did
the affected brother also carry the mutation. Germ-line mutations in
BRCA2 may therefore account for about 5% of prostate
cancer in familial clusters.

INTRODUCTION

Prostate cancer is the second most common cause of cancer
mortality in men in the United Kingdom. Approximately 14,000 cases/year
and 8,742 deaths/year are reported in England and Wales
(1, 2)
. Its incidence is increasing by 10% every 5 years
(3)
, even when the effect of screening is taken into
account, and 13% of cases occur in men in their preretirement years.
One percent of cases occur in men <55 years of age in the
United Kingdom. There is increasing evidence that there is an inherited
component to many of the common cancers
(4)
, and prostate
cancer is no exception. Familial clustering of prostate cancer has been
observed, the most dramatic of which is the large prostate cancer
kindreds described in Utah, United States
(5)
;
furthermore, case-control studies
(6)
show that relatives
of cases have an increased relative risk of developing the disease, and
this has been confirmed in two cohort studies
(7, 8)
. This
relative risk increases markedly when the age of the index case
decreases or the number of affected individuals in a cluster increases,
which is evidence that this increased risk has a genetic component. One
segregation analysis has led to the proposed model of at least one
highly penetrant gene (88% of gene carriers would develop prostate
cancer by age 85) that accounts for 43% of cases diagnosed at <55
years
(9)
; two others support this model, but with a
higher gene frequency of about 1% and a penetrance of 63%
(10, 11)
. Other reports have suggested a recessive or X-linked model
(12, 13)
. Recently, a highly penetrant prostate cancer
susceptibility locus HPC1 was mapped to 1q, but this only
accounts for up to 34% of families with four or more cases in one
study of 91 families
(14)
or, at most, 20% of such large
clusters in another study of 35 families
(15)
. In the
latter study, 1q linkage analysis in 101 clusters with ≤3 cases showed
no evidence of linkage to 1q. Recent studies have suggested that other
loci exist: (a) one at 1q42
(16)
that has not
yet been confirmed; (b) one at Xq27–28
(17)
that accounts for 16% of families; and (c) one at 1p36
(18)
. Preliminary evidence from analysis of 187 prostate
cancer clusters in our laboratory indicates that these loci do not
account for all of familial prostate cancer. Other genes therefore
remain to be located.

There is an association between breast and prostate cancer in families;
a higher incidence of prostate cancer among male relatives of breast
cancer patients has been reported previously
(19,
20,
21)
.
Anderson and Badzioch
(22)
report a doubling of familial
breast cancer risk when prostate cancer is present in the family
history. BRCA1 and BRCA2 are located on
chromosomes 17q12–21 and 13q12–13, respectively.
LOH
4
studies in prostate cancer have shown that 52% of tumors have LOH at
17q; in one study, 44% of tumors had LOH with a marker intragenic in
the breast cancer predisposition gene BRCA1(23)
. BRCA1 carriers also have a 3-fold
increased risk of mortality from prostate cancer
(24)
. We
have demonstrated a 25% incidence of LOH at the BRCA2 locus
in familial and sporadic prostate cancer
(25)
. Tonin
et al.(26)
calculated that there was a
relative risk of 7.2 of prostate cancer in BRCA2 carriers
but did not mention an age-at-onset effect.

One family with four prostate cancer cases but no breast cancer has
been reported to have a germ-line BRCA1 mutation
(27)
that is 185delAG, a common BRCA1 mutation
in Ashkenazi Jewish families with breast cancer
(28)
. This
family was indeed of this ethnic origin. Workers from Iceland
(29)
have reported a common BRCA2 mutation
(999del5) in nine Icelandic cancer families with multiple cases of
breast cancer. Some of these families also had multiple cases of
prostate cancer. Icelandic studies have shown 2.7% of prostate cancer
cases in Iceland carry this mutation
(30)
. Four studies
(31,
32,
33,
34)
have reported that there is no increased
frequency of the founder Ashkenazi BRCA1 and
BRCA2 mutations over that expected in this population when
germ-line DNA from prostate cancer cases with and without a family
history are analyzed.

The Cancer Research Campaign/British Prostate Group United Kingdom
Familial Prostate Cancer Study aims to investigate the role of genetic
susceptibility to prostate cancer. As part of the study of high
penetrance genes, prostate cancer cases with an increased chance of
harboring a prostate cancer susceptibility gene are being collected.
Those clusters with a relative risk of developing prostate cancer of≥
4 are targeted for collection
(35)
; these are clusters
of ≥3 prostate cancers at any age or in sibling pairs, preferably
where one is <65 years at diagnosis. The first 38 of these
clusters were analyzed in this study; BRCA1 and
BRCA2 were analyzed from germ-line DNA to assess the
contribution of BRCA1 and BRCA2 germ-line
mutations to familial prostate cancer. This is the first study to
analyze the entire coding region of BRCA1 and
BRCA2 in a non-Ashkenazi series of prostate cancer clusters.

MATERIALS AND METHODS

Patient Material.

Peripheral blood DNA from 38 affected individuals who were members of
prostate cancer clusters was studied. The composition of the clusters
is shown in Table 1
⇓
. Wherever possible, DNA from the youngest available member of each
cluster was studied. Tumor DNA was prepared after microdissection of
tumor tissue from paraffin sections. Microdissected tissue was removed
into 200 μl of extraction buffer [1× RedHot polymerase buffer
(Applied Biosystems), 1.5 m
MgCl2, 0.45% NP40, 0.45% Tween 20, and 200μ
g/ml proteinase K] and incubated at 55°C for 12 h.
After incubation, proteinase K was deactivated by heating to 99°C for
10 min.

Mutation Analysis.

BRCA1 and BRCA2 were both screened for
germ-line mutations using a combination of the PTT and a nonradioactive
HA to identify variants in the sample set. PTT is an efficient
technique for screening large DNA fragments (≥1 kb) for truncating
mutations and was used to analyze exon 11 of BRCA1 (which
represents approximately 60% of the coding sequence) and exons 10 and
11 of BRCA2 (60% of the coding region). The remaining exons
and splice boundaries of both genes were screened using HA. The
majority of germ-line mutations reported in BRCA1 and
BRCA2 result in truncation of the predicted protein as a
result of frameshift, nonsense, or splice site alterations; therefore,
the combination of PTT and HA was considered a sensitive and efficient
method of analysis. Direct sequence analysis was used to confirm the
precise nucleotide alteration associated with PTT and/or HA variants.
The primer sequences for BRCA2 and their respective product sizes and
amplification conditions have been described previously
(36)
.

PTT was performed for the largest two exons of BRCA2 and for
the largest exon only for BRCA1. Primers were designed to
PCR amplify exons 10 and 11 of BRCA2 and exon 11 of
BRCA1 from genomic DNA in overlapping fragments ranging in
size from 1.0–1.3 kb. PTT was performed as described previously
(36)
.

Coding exons 2, 3, 5–10, and 12–24 of BRCA1 and 2–9 and
12–27 of BRCA2 were amplified from genomic DNA. The 5′ and
3′ splice boundaries for exon 11 of BRCA1 and exons 10 and
11 of BRCA2 were also amplified from genomic DNA.
SSCA/HA was performed in 1× mutation detection
enhancement polyacrylamide gels as described previously
(36)
. Syder Green staining was used for DNA detection.
Sequence analysis of variant PTT and SSCA/HA samples was
performed using the ABI 373A DNA sequencer by dye terminator cycle
sequencing with AmpliTaq DNA polymerase FS (Perkin-Elmer).

Haplotype Analysis.

Peripheral blood DNA and tumor DNA from paraffin-embedded tumor
tissue was PCR amplified with three polymorphic microsatellite markers,
D13S260, D13S263, and D13S267, which flank the BRCA2 gene on
chromosome 13q12. PCR products were electrophoresed at 250 V on 8–12%
polyacrylamide gels for 14–16 h at a constant temperature of 18°C.
Gels were visualized after silver staining as described previously
(36)
.

Immunohistochemical Staining for BRCA2 Protein.

Sections (4 μm) were cut from blocks of prostate cancer tissue,
picked up on adhesive-coated slides (Vector Laboratories, Burlingame,
CA), and baked overnight at 56°C before staining. The BRCA2 antigen
was unmasked by placing the sections in a pressure cooker containing
boiling 0.01 m citrate buffer (pH 6.0) and boiling
under pressure for 2 min. Sections were cooled in running tap water and
rinsed in Tris-buffered saline before the application of the rabbit
polyclonal BRCA2 antibody (courtesy of N. Spurr and D. M. Barnes,
Imperial Cancer Research Fund) for 1 h. Antibody binding
was detected using a conventional peroxidase-conjugated streptavidin
biotin complex method (Dako Ltd., High Wycombe, United Kingdom). Sites
of peroxidase activity were detected using diaminobenzidine as the
chromogen. A breast carcinoma known to express BRCA2 was used as a
positive control. A negative control in which the primary antibody was
replaced with Tris-buffered saline was included for each case. The
presence of any nuclear staining was recorded as positive.

The BRCA2 antibody used was raised against a COOH terminus peptide
synthesized using the sequence published by Wooster et al.(37)
. The peptide, which contained residues 2301–2320
(DGKGKEEFYRALCDVKAT) with a peak corresponding to a calculated
Mr of 2101, was prepared using
the fastmoc HBTU method to a standard purity
(25)
.

RESULTS

Mutation analysis of BRCA1 revealed no variants that
appeared to be related to the disease phenotype in any of the 38
prostate cancer families. Several frequently observed variants were
detected using HA, but sequencing revealed these to be either coding or
noncoding polymorphisms that have been reported
previously.
5

Analysis of BRCA2 revealed three variants that were
not detected in any other individuals from the sample set. Two of these
variants were detected as truncated proteins by PTT; the third was
detected as a heteroduplex variant in exon 22. In family PRY1042, from
individual 201, a PTT variant in exon 11 (Fig. 1
⇓
) was characterized as a 4-bp deletion beginning at nucleotide 6710
(6710delACAA) that is predicted to cause a frameshift and premature
truncation of the predicted protein at codon 2166. This mutation has
not been reported previously. HA using primers designed to amplify the
region flanking this mutation confirmed the presence of this alteration
in the index case and also showed loss of the wild-type allele in DNA
from tumor tissue from the same individual. However, HA of DNA prepared
from tumor tissue from the affected sibling, who was diagnosed with
prostate cancer at 48 years, indicated that this individual did not
carry the BRCA2 mutation (Fig. 2a⇓
). Haplotype analysis using three polymorphic microsatellite
markers flanking the BRCA2 gene at chromosome 13q12–13 was
performed on DNA from the two affected brothers from family PRY1042.
Although it was not possible to phase the haplotypes, the allele sizes
of each marker indicate that both copies of chromosome 13 differ
between the two brothers (data not shown). This is consistent with the
observation of a germ-line BRCA2 mutation in one affected
brother but not in the other.

a, the pedigree of family PRY1042. The
mutation in this family, 6710delACAA, is detected as a heteroduplex
variant (het) in index case 201. This variant is not
present in the affected brother (202) or in a normal
sample (N). pa ca, pancreatic cancer;
pr ca, prostate cancer. Analysis of tumor DNA from
individual 201 shows loss of the wild-type homoduplex DNA band
(wt-hd) and retention of the shorter, mutant homoduplex
band (m-hd). This is compared with wild-type homoduplex
DNA seen in a normal sample (N). b, the
mutation in family PRS2024, 5531delTT, was detected as a heteroduplex
variant (het) and a homoduplex conformer
(hd) in blood DNA from index case 201. This variant is
not present in blood DNA from the affected father (101)
or in tumor DNA (T) from the affected brother
(202). br ca, breast cancer.

A PTT variant in exon 11 in a prostate cancer case diagnosed at 56
years from family PRS2024 (Fig. 1
⇓
) was characterized as a 2-bp deletion
beginning at nucleotide 5531 (5531delTT). This is a novel mutation and
is predicted to result in frameshift and truncation at codon 1772. The
family history of PRS2024 with respect to the index case consists of
the father diagnosed with prostate cancer at 87 years, the mother
diagnosed with breast cancer at 76 years, and a brother diagnosed with
prostate cancer at 41 years. HA confirmed the presence of the mutation
in the index case but indicates that the same alteration is not present
in the father’s constitutional DNA or in tumor DNA from the affected
brother (Fig. 2b⇓
). No DNA was available from the proband’s
mother. Neither deletion was found in over 100 normal
individuals tested.

A heteroduplex variant was detected in exon 22 in a prostate
cancer case diagnosed at 46 years, from family PRS1081. This variant
was characterized as a single-base substitution (G to T at nucleotide
9078) that is predicted to convert a lysine amino acid residue to an
asparagine residue (K2950N) and has not been reported previously. DNA
was not available from a second affected individual from the family to
confirm segregation of this alteration with the disease. To examine
whether this alteration is a putative missense mutation or merely a
rare variant without disease association, DNA was analyzed from a
series of normal individuals for the presence of the sequence change.
The identification of the K2950N alteration in 2 of 340 (0.59%) normal
chromosomes suggests that this change is a rare polymorphism that is
not associated with the disease. Several other heteroduplex variants
were observed throughout BRCA2 in the sample set. However,
these all occurred relatively frequently and were characterized as
either previously reported coding or intronic polymorphisms that are
not considered to be disease related.

The BRCA2 antibody stains 25%
(25)
of sporadic
prostate cancer samples. We found that the two individuals with
deletions in BRCA2 did not exhibit any staining in their
prostate tumors, but their siblings without mutation and the individual
with the K2950N variant did so. In the latter case, multifocal areas of
intense nuclear staining were observed within tumor areas (Fig. 3 and b⇓
).

a, prostate cancer cells from the brother
(PRY1042.202) of the individual (PRY1042.201) who has a germ-line
deletion (6710delACAA). This shows weakly positive staining with
antibody to BRCA2 protein in individual 1042.202, who does not carry
the mutation. (×400). b, multifocal areas of intense
staining for antibody to BRCA2 protein in prostate cancer cells from
the patient who has a polymorphism in BRCA2 (K2950N variant; ×400).

DISCUSSION

The data we have reported suggest that approximately 5% (2
of 38) of families identified with a history of prostate cancer, based
on either affected sibling pairs or three or more affected individuals
in the family, may contain an individual with a germ-line mutation in
the BRCA2 gene. These data also indicate that
BRCA1 does not contribute significantly to familial prostate
cancer. The actual proportions of germ-line BRCA2 and
BRCA1 mutations in such families may be greater; it is
possible that mutations may have been missed using the combination of
the PTT and HA, and we would not have detected missense mutations in
the regions screened by PTT. It is surprising that the two
disease-associated BRCA2 mutations that were detected were
not present in the affected sibling in each of the families. In family
PRY1042, the germ-line mutation 6710delACAA and loss of the wild-type
allele in tumor tissue detected in the index case suggest that the
mutation in BRCA2 is cancer-causing and acting as a tumor
suppressor gene. This is consistent with previous reports that suggest
that sporadic ovarian cancers with germ-line BRCA2 mutations
and breast tumors from a BRCA2 linked family show nonrandom
loss of the wild-type allele
(38, 39)
. The germ-line
mutation in family PRS2024 was detected in the index case diagnosed
with prostate cancer but not in his father (who was diagnosed at 87
years), suggesting that the father may be a sporadic case, nor was it
present in tumor DNA from the affected brother. The mother was
diagnosed with breast cancer at 76 years of age, and it is probable
that she is also a germ-line carrier of the mutation, although no DNA
was available to test this hypothesis.

The fact that several reports have now shown that germ-line
mutations in the BRCA2 gene are associated with an increased
risk of prostate cancer
(29, 40, 41)
makes
BRCA2 a putative candidate gene for familial prostate cancer
in general. Our data using linkage analysis at the BRCA2
locus in 100 affected sibling pairs with prostate cancer has estimated
that up to 30% of such pairs (95% confidence interval, 0–70%) may
be due to the BRCA2 gene
(42)
. Although the
Cancer Research Campaign/British Prostate Group United Kingdom Familial
Prostate Cancer Study ascertained sibling pairs with at least one of
the affected siblings at age <67 years at diagnosis, the two mutations
described here have occurred in prostate cancer cases occurring at ≤56
years, and BRCA2 germ-line mutations may therefore
contribute to a significant proportion of young cases within these
pairs. We were surprised to find that in both of the sibling pairs, the
BRCA2 mutation was not present in the affected brother. This
was unexpected because both brothers affected who did not carry the
mutation were younger than those who did. There are two possible
explanations for this. The first is that the BRCA2 mutation
is not cancer causing. This is unlikely because both mutations are
deletions and would be expected to have a major effect on the function
of the protein. Furthermore, the wild-type allele was lost in the
subsequent prostate tumor in PRY1042, individual 201. It is interesting
that BRCA2 antibody staining was negative in both patients
with BRCA2 germ-line mutations but positive in their
brothers who did not have a truncating mutation and also in the
individual with the K2950N variant. The overall frequency of positive
BRCA2 staining in sporadic prostate cancer is 25%
(25)
. The second possible explanation is that there is
another gene segregating in the prostate cancer clusters PRY1042 and
PRS2024 and that the BRCA2 mutation is acting as a modifier.
There is some evidence for this in Icelandic families with
BRCA2 mutations, in which prostate cancer incidence is
inversely proportional to male breast cancer incidence in branches of
the same family with the same germ-line mutation
(29)
.
Both families with BRCA2 mutations contained an individual
with a cancer at another site. PRY1042 contained a case of pancreatic
cancer, and PRS2024 contained a case of breast cancer. In PRS2024, the
father of the prostate cancer case with the germ-line BRCA2
mutation did not have the mutation, despite being affected with the
disease. If it had been inherited and was not a novel mutation, then
this is presumed to have been inherited from the case’s mother. This
raises the possibility that BRCA2 germ-line mutations are
only seen in the context of prostate cancer families with associated
cancers known to occur in BRCA2 families [namely breast,
pancreatic, ovarian, and gall bladder cancer
(41)]
. Of
the 38 families, 25 (66%) had prostate cancer and other cancers. Table 1
⇓
lists the other cancers. Of 25 prostate cancer families with prostate
and other cancers, two had germ-line mutations in BRCA2
(8%). None of the 13 families with prostate cancer alone had
BRCA2 mutations. Our data suggest that a proportion of
prostate cancer families may harbor germ-line mutations in the
BRCA2 gene. Because the clusters we analyzed were small, it
is possible that we have underestimated the contribution of germ-line
mutations in BRCA2 to prostate cancer overall. Additional
studies are warranted in larger series of both prostate cancer clusters
and isolated cases at varying ages to determine the size of this
proportion in different prostate cancer populations.

Acknowledgments

The contribution of all of the members of the families in
this study is gratefully acknowledged. We are grateful to S. Osborne
for data management.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported by the Cancer Research Campaign; The
Institute of Cancer Research, Prostate Cancer Research, United Kingdom;
and Imperial Cancer Research Fund. S. M. E. was supported by Royal
Marsden National Health Service Trust Charitable Funds and is
now supported by the Cancer Research Campaign. D. P. D. is supported
by the Bob Champion Cancer Trust, and W. D. D. is supported by
Zeneca, Yamanouchi, and Merck Sharpe & Dohme Pharmaceuticals.
B. A. J. P. is a Gibb Fellow of the Cancer Research Campaign.

↵2 The Cancer Research Campaign/British Prostate
Group United Kingdom Familial Prostate Cancer Study
collaborators. List available on request.